Device and method for monitoring leukocyte migration

ABSTRACT

A device for monitoring leukocyte migration is provided. The invention also provides a method of using the device to monitor leukocyte migration in the presence of physiological shear flow and therefore simulate physiological conditions of a blood vessel in vivo. The invention further provides a method of using the device to high-throughput screen a plurality of test agents. The present invention further provides a flexible assay system and numerous assays that can be used to test biological interactions and systems. Laminar flow gradients are employed that mimic gradient situations present in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. applicationSer. No. 10/241,445 (filed Sep. 12, 2002), which is acontinuation-in-part of U.S. patent application Ser. No. 10/097,329(filed Mar. 15, 2002), U.S. patent application Ser. No. 10/097,351(filed Mar. 15, 2002 now U.S. Pat. No. 6,921,660), U.S. patentapplication Ser. No. 10/097,306 (filed Mar. 15, 2002), U.S. patentapplication Ser. No. 10/097,304 (filed Mar. 15, 2002 now U.S. Pat. No.6,818,403), U.S. patent application Ser. No. 10/097,322 (filed Mar. 15,2002 now U.S. Pat. No. 6,811,968), U.S. patent application Ser. No.10/097,302 (filed Mar. 15, 2002), U.S. patent application Ser. No.09/709,776 (filed Nov. 8, 2000 now U.S. Pat. No. 6,699,665), and U.S.patent application Ser. No. 10/206,112 (filed Jul. 29, 2002 now U.S.Pat. No. 6,893,851). The present application also claims the benefit ofU.S. Provisional Applications Nos. 60/419,980 and 60/419,976 filed onOct. 22, 2002, the contents of which are incorporated by referenceherein.

FIELD OF INVENTION

The present invention relates to devices and methods for monitoring theinteraction of a cell or group of cells with a substratum. Inparticular, the present invention relates to devices and methods formonitoring leukocyte migration. The present invention also relatesgenerally to biological assays performed in gradients formed inmicrofluidic systems.

BACKGROUND

The inflammatory response is an attempt by the body to restore andmaintain homeostasis after infection or injury, and is an integral partof body defense. Most of the body defense elements are located in theblood and inflammation is the means by which these elements leave theblood and enter the tissue around the injured or infected site. Theprimary objective of inflammation is to localize and eradicate thesource of injury or infection and repair tissue surrounding the site ofinjury or infection.

As a consequence of the initial innate immune response to infection,phagocytes such as mast cells in the damaged tissue release a variety ofcytokines and inflammatory mediators, such as histamines, leukotrienes,bradykinins, and prostaglandins. These inflammatory mediators reversiblyopen the junctional zones between the thin delicate cells of the innersurface of the blood vessels, known as the endothelium, that surroundthe damaged tissue. The inflammatory mediators also cause increasedblood vessel permeability and decreased blood flow velocity. Anotherresult of these changes in the blood vessels is that leukocytes, whichnormally travel in the center of the blood vessel, move out to theperiphery of the inner surface of the blood vessel to interact with theendothelium. The cytokines and inflammatory mediators released by thephagocytes also induce the expression of adhesion molecules on thesurface of the endothelium, resulting in an “activated” endothelium.

The first contact of leukocytes with the activated endothelium is knownas “capture” and is thought to involve the adhesion molecules P-selectinand L-selectin, which are upregulated on endothelium after exposure toinflammatory mediators. P-selectin and L-selectin belong to a family ofadhesion molecules called selectins. Selectins are a group of monomeric,integral membrane glycoproteins expressed on the surface of activatedendothelium and leukocytes. Selectins contain an N-terminalextracellular domain with structural homology to calcium-dependentlectins, followed by a domain homologous to epidermal growth factor, andnine consensus repeats (CR) similar to sequences found in complementregulatory proteins. There are three primary selectins thought to beinvolved in the inflammatory response: P-selectin; E-selectin; andL-selectin. P-selectin, also known as CD62P, GMP-140, and PADGEM, thelargest selectin, is expressed on activated endothelium; E-selectin,also known as ELAM-1, is expressed on endothelium with chemically orcytokine-induced inflammation; L-selectin, also known as LECAM-1, LAM-1,Mel-14 antigen, gp90^(mel), and Leu8/TQ-1 antigen, is the smallestselectin and is found on most leukocytes. All three selectins arethought to bind to selectin binding ligands, at least in part through acarbohydrate component.

During capture, P-selectin is thought to bind to its main leukocyteligand P-selectin glycoprotein ligand-1 (PSGL-1). Other ligands ofP-selectin include CD24 and yet uncharacterized ligands. The structureof functional PSGL-1 includes a sialyl-Lewis^(x) component. In addition,during capture L-selectin is thought to bind to its ligand onendothelial cells. L-selectin interacts with three known counterreceptors or ligands, MAdCAM-1, GlyCAM-1, and CD34, although the preciseligand or counter receptor involved in capture is unknown.

Once leukocytes are captured, they may transiently adhere to theendothelium and begin to “roll” along the endothelium. The term“rolling” refers to the literal rolling of leukocytes along theactivated endothelium in the presence of fluid drag forces arising fromthe relative movement between the endothelium and the leukocytes.Rolling is thought to involve P-selectin, L-selectin, and E-selectin.Bonds between P-selectin and PSGL-1 are thought to primarily mediate the“rolling” of leukocytes across the endothelium.

Proinflammatory cytokines such as interleukin-1 (IL-1), and tumornecrosis factor-α (TNF-α) produced by cells at the injured or infectedsite stimulate the endothelium to produce chemokines such asinterleukin-8 (IL-8) and integrin binding ligands such as intercellularadhesion molecules (ICAMs) and vascular cell adhesion molecules (VCAMs)on the surface of the endothelial cells opposite the basal lamina. Thechemokines are held on the surface of the endothelial cells opposite thebasal lamina where the chemokines interact with chemokine receptors onthe surface of the rolling leukocytes. This interaction, in turn,triggers the activation of molecules called integrins on the surface ofthe leukocytes. Integrins are a family of heterodimeric transmembraneglycoproteins that attach cells to extracellular matrix proteins of thebasement membrane or to ligands on other cells. Integrins are composedof large α and small β subunits. Mammalian integrins form severalsubfamilies sharing common β subunits that associate with different αsubunits. ∃₂ integrins (the “CD-18 family”) include four differentheterodimers: CD11a/CD18 (Lymphocyte Function-Associated Antigen-1(LFA-1)); CD11b/CD18 (Mac-1); CD11c/CD18 (p150,95), and CD11d/CD18. Themost important member of the ∃₁ integrin subfamily on leukocytes is VeryLate Antigen 4 (VLA-4, CD49d/CD29, ∀₄∃₁). Activation of these integrinsby chemokines enables the slowly rolling leukocytes to “arrest” andstrongly bind to the endothelium's ICAMs, VCAMs, and other integrinbinding ligands of the endothelial cells, such as collagen, fibronectin,and fibrinogen. Once bound to the endothelial cells, the leukocytes thenflatten and squeeze between the endothelial cells to leave the bloodvessels and enter the damaged tissue through a process termed“transmigration.” Transmigration is thought to be mediated by platelets,endothelial cell adhesion molecule-1 (PECAM-1), junctional adhesionmolecule (JAM), and possibly CD99, a transmembrane protein.

Despite their importance in fighting infection and injury, leukocytesthemselves can promote tissue damage. During an abnormal inflammatoryresponse, leukocytes can cause significant tissue damage by releasingtoxic substances at the vascular wall or in uninjured tissue.Alternatively, leukocytes may stick to the capillary wall or clump invenules to such a degree that the endothelium becomes lined with thesecells. Such a phenomenon, referred to as “pavementing,” may be relatedto the development of arteriosclerosis and associated diseases. Suchabnormal inflammatory responses have been implicated in the pathogenesisof a variety of other clinical disorders including adult respiratorydistress syndrome (ARDS); ischemia-reperfusion injury followingmyocardial infarction, shock, stroke, or organ transplantation; acuteand chronic allograft rejection; vasculitis; sepsis; rheumotoidarthritis; and inflammatory skin diseases.

Several methods and devices exist in the art to study the processes ofleukocyte migration implicated in these various inflammatory diseases.For example, one method involves plating a monolayer of isolatedendothelial cells on the surface of microtiter plates, activating thecells with a chemoattractant and then placing labeled leukocytes in theplate. A test agent, such as an adhesion inhibitor, may be optionallyadded to the plate. The number of leukocytes that remain adherent to theendothelial cell monolayer is then determined. A significantdisadvantage of this method is that the leukocytes are not exposed tothe endothelial cells in the presence of shear flow and thus this methoddoes not simulate physiological conditions in vivo.

Another method involves contacting a suspension of isolated leukocytesin a suitable medium with a human vascular tissue sample mounted on amicroscope slide and then incubating the tissue with a cell suspensionon a rotating table. The adhered cells are fixed and counted. Becausecells are fixed, such a method precludes the observation of leukocytemigration in real time. In addition, such a method requires humanvascular tissue, which can be difficult and costly to obtain.

Another method known in the art to study leukocyte migration, involves adevice consisting of two glass tubes called microslides, one microslidecapable of being inserted into the other. The smaller microslide isinserted into the larger one to create a flow channel with a flatsurface on which selected adhesion molecules are present. A suspensionof leukocytes is then perfused through the flow channel over theadhesion molecule immobilized surface using a syringe pump. The rollingand adhesion of the leukocytes is then observed. Because of the size andconfiguration of this device, it requires considerable handling andmanipulation.

Another device to study leukocyte migration during the inflammatoryresponse is described in U.S. Pat. No. 5,460,945 to Springer et al.entitled “Device and Method for Analysis of Blood Components andIdentifying Inhibitors and Promoters of the Inflammatory Response.” Thisdevice consists of several different components that are bulky in size.As such, it requires extra handling and positioning, creating the riskof contaminating or damaging the endothelial monolayer. This device alsorequires the use of a large number of cells and consequently a largeamount of reagents.

Therefore, there exists a need for an improved device to study theleukocyte migration along the endothelium that simulates thephysiological conditions of a blood vessel. There also exists a need fora device that would allow for high throughput screening of test agentsthat potentially affect the interaction of leukocytes with theendothelium without requiring the number of leukocytes per assay asrequired by the devices currently known in the art. The presentinvention meets these needs.

SUMMARY OF THE INVENTION

The present invention provides a system for monitoring leukocytemigration comprising a device including a housing defining a pluralityof chambers therein. Each of the plurality of chambers includes a firstwell region including at least one first well, a second well regionincluding at least one second well, and a channel region including atleast one channel connecting the first well region and the second wellregion with one another. The system further includes a first fluidstream having a first concentration of a first substance and a secondfluid stream having a second concentration of a second substance,wherein the first and second concentrations are different from oneanother and the first and second fluid streams are in fluidcommunication with at least one of the plurality of chambers.

The present invention also provides a method of monitoring leukocytemigration comprising disposing endothelial cells on a surface, passing afluid along the surface under conditions of substantially laminar flowwherein the fluid comprises a concentration gradient of at least onesubstance. The concentration gradient is substantially perpendicular tothe direction of flow. The method further includes exposing a samplecomprising leukocytes to the surface, and observing the interactionbetween the leukocytes and the endothelial cells.

The present invention moreover provides a method of monitoring leukocytemigration comprising providing a device including a housing defining aplurality of chambers therein. Each of the plurality of chambersincludes a first well region including at least one first well, a secondwell region including at least one second well, and a channel regionincluding at least one channel connecting the first well region and thesecond well region with one another. The method further comprisesdisposing at least one leukocyte migration mediator, or endothelialcells in the at least one channel, delivering a sample comprisingleukocytes to the at least one channel by laminar flow, and observingthe interaction between the leukocytes and the at least one leukocytemigration mediator or the interaction between the leukocytes and theendothelial cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description given hereinbelow and the accompanying drawingswhich are given by way of illustration only, and thus are not limitativeof the present invention, and wherein:

FIG. 1 is a perspective view of an embodiment of a device adapted to beused in a method for monitoring leukocyte migration according to thepresent invention.

FIG. 2 is a cross-sectional view of the device of FIG. 1 along linesII-II.

FIG. 3 is a top plan view of the device of FIG. 1.

FIG. 4 is a top plan view of an alternative embodiment of a chamberdefined in a housing of a device adapted to be used in a method formonitoring leukocyte migration according to the present invention.

FIG. 5 is a top plan view of a plurality of chambers such as the chamberof FIG. 4 disposed in a predetermined relationship with respect to oneanother.

FIG. 6 is a top, perspective view of an alternative embodiment of adevice adapted to be used in a method for monitoring leukocyte migrationaccording to the present invention, where the device displays thedimensions and pitch of a standard 96-well microtiter plate.

FIG. 6A is a top enlarged view of an individual well of an alternativeembodiment of the device according to the present invention.

FIG. 7 is a bar graph comparing the velocity of shear flow underdifferent cell suspension volumes.

FIG. 8 is a graph comparing the number of cells rolling under differentdilutions of P-selectin antibody.

FIG. 9 is a graph and time-lapsed still photographs of cells rolling andadhering under different dilutions of P-selectin antibody.

FIG. 10 is a graph and time-lapsed still photographs of cells rollingand adhering under different dilutions of E-selectin antibody.

FIG. 11 is a graph and time-lapsed still photographs of cells adheringto endothelium in the presence of antibodies to E-selectin, P-selectin,and VCAM-1.

FIG. 12 depicts the results of an experiment involving the creation of aconcentration gradient of TNF-α via laminar flow. The TNF-α wasdelivered to a confluent “lawn” of endothelial cells. The endothelialcells that were contacted by the TNF-α were activated and thus are ableto bind the leukocytes. Leukocytes were then delivered to theendothelial cells. As is demonstrated in the figure, the leukocytesbound to the area of the endothelial cells that received highconcentrations of TNF-α whereas those areas not exposed to TNF-α orexposed to very little TNF-α did not bind leukocytes.

FIG. 13 depicts an exemplary microfluidic device for creating a laminarflow gradient.

DETAILED DESCRIPTION

Definitions

It is understood that the terminology and definitions used herein arefor the purpose of describing particular embodiments only and are notintended to be limiting.

The term “leukocytes” as used herein refers to granulocytes includingneutrophils, eosinophils, basophils, monocytes, and lymphocytesincluding B cells and T cells and unless otherwise specified, platelets.The term “leukocytes” includes leukocytes obtained from both normalblood samples and pathological blood samples.

The term “leukocyte migration cascade” refers to the cascade ofsequential events involving a leukocyte's migration along theendothelium lining a blood vessel. The leukocyte migration cascadeincludes the capture, rolling, arrest, and transmigration of a leukocyteon, along, or through the endothelium.

The term “leukocyte migration mediator” as used herein refers to anymolecule that mediates the migration of leukocytes along the endotheliumlining a blood vessel. The term “mediates” as used in the context of a“leukocyte migration mediator” means influencing the migration of aleukocyte by, for example, binding to the ligand or counter-receptor ofthe leukocyte migration mediator. In particular, the term “leukocytemigration mediator” refers to any molecule involved in the leukocytemigration cascade. As such, a leukocyte migration mediator includes aleukocyte capture mediator, a leukocyte rolling mediator, a leukocytearrest mediator, a leukocyte transmigration mediator, or any combinationthereof.

The term “capture” as used herein refers to a step in the leukocytemigration cascade characterized by the tethering or first contact ofleukocyte with the endothelium of a blood vessel so that the motion ofthe leukocyte along the endothelium is temporarily delayed relative tothe flow of fluid containing free flowing leukocytes.

The term “leukocyte capture mediator” as used herein refers to aleukocyte migration mediator that mediates the capture of a leukocyte onthe endothelium of a blood vessel. Non-limiting examples of leukocytecapture mediators are P-selectin and L-selectin binding ligands.

The term “capture mediator binding partner” refers to any ligand orcounter-receptor that binds a leukocyte capture mediator. Non-limitingexamples of capture mediator binding partners are PSGL-1 and L-selectin.

The term “rolling” as used herein refers to a step in the leukocytemigration cascade, and is characterized by the rolling of a leukocytealong the endothelium of a blood vessel from receptor to receptor on theendothelium further characterized by leukocytes forming and breakingadhesive bonds with endothelial ligands or counter-receptors.

The term “leukocyte rolling mediator” as used herein refers to anyleukocyte migration mediator that mediates the rolling of a leukocytealong the endothelium of a blood vessel. Non-limiting examples ofleukocyte rolling mediators are P-selectin, E-selectin, and L-selectinbinding ligands.

The term “rolling mediator binding partner” as used herein refers to anyligand or counter-receptor that binds to a leukocyte rolling mediator.Non-limiting examples of rolling mediator binding partners are PSGL-1,E-selectin binding ligand, and L-selectin.

The term “arrest” as used herein refers to a step in the leukocytemigration cascade characterized by the adherence of leukocytes to theendothelium of a blood vessel.

The term “leukocyte arrest mediator” as used herein refers to anyleukocyte migration mediator that mediates the arrest of a leukocyte onthe endothelium of a blood vessel. Non-limiting examples of arrestmediators are integrin binding ligands, such as ICAM-1, ICAM-2, andVCAM-1 that bind integrins expressed on the surface of leukocytes.

The term “arrest mediator binding partner” as used herein refers to anyligand or counter-receptor that binds to a leukocyte arrest mediator.Non-limiting examples of arrest mediator binding partners are integrinsincluding LFA-1, Mac-1, p150,95, VLA-4, and VLA-5.

The term “transmigration” as used herein refers to a step in theleukocyte migration cascade characterized by the exit of leukocytes froma blood vessel to surrounding tissue through passage between cells ofthe endothelium of the blood vessel.

The term “leukocyte transmigration mediator” as used herein refers toany leukocyte migration mediator that mediates the transmigration of aleukocyte through the endothelium of a blood vessel. Non-limitingexamples of leukocyte transmigration mediators are PECAM-1 and JAM.

The term “transmigration binding partner” as used herein refers to anyligand or counter-receptor that binds to a leukocyte transmigrationmediator.

The term “physiological shear flow” includes shear flow under normal andpathological conditions. Physiological shear flow rate under normalconditions is about 0.1 to about 20 dynes/cm².

The term “test agent” as used herein refers to any chemical molecule orcompound to be tested in the present invention to determine its abilityto interact with another chemical molecule or compound. For example, thetest agent may be tested to determine whether it inhibits or promotesleukocyte migration by inhibiting or promoting capture, rolling, arrest,or transmigration.

The term “pitch” as used herein refers to the distance betweenrespective vertical centerlines between adjacent wells in the testorientation of the device.

The term “well region” as used herein is meant to refer to a region thatcomprises one or a plurality of wells.

The term “well” as used herein is meant to indicate any cavity that isable to receive a fluid therein.

The term “channel region” as used herein refers to a region thatcomprises one or a plurality of channels therein, while “channel” refersto any passageway.

In the context of the present invention, “conformal contact” is meant todesignate a substantially fluid-tight, form-fitting contact with aplanar or non-planar surface, and “reversible conformal contact” ismeant to designate a conformal contact that may be interrupted withoutcompromising a structural integrity of the members making the conformalcontact.

In the context of the present invention, the “test orientation” of thedevice is meant to refer to a spatial orientation of the device duringthe monitoring of leukocyte migration and the terms “top,” “bottom,”“upper” and “side” are defined relative to the test orientation of thedevice. In one embodiment, the test orientation of the device for use ina method of monitoring leukocyte migration contemplates the orientationof the device such that a migration path along the channel region of anycells occurs in a substantially horizontal plane. In another embodiment,the test orientation of the device for use in monitoring leukocytemigration contemplates the orientation of the device such that amigration path along the channel region of any cells occurs in asubstantially vertical plane.

The present invention generally provides devices and methods for invitro monitoring the interaction of cells with a substratum.Non-limiting examples of cell types that may be monitored by the devicesand methods of the present invention include leukocytes, red bloodcells, platelets, non-blood cells, and tumor cells. Non-limitingexamples of types of substratum that may interact with the cells includethe endothelium, immobilized ligands, physisorbed adhesion and rollingmolecules and basal lamina or basal lamina mimic. In particular, thepresent invention provides a device and method for in vitro monitoringof leukocyte migration in the presence of shear flow in order to studythe cascade of events involved in the inflammatory response in vivo. Thepresent invention also provides a device and method for thehigh-throughput screening of test agents that potentially target theseevents. In particular, the present invention is directed to study andtarget the capture, rolling, arrest, and transmigration of a leukocyteon, along, or through the endothelium (such events collectively referredto as the “leukocyte migration cascade”).

As schematically depicted in FIGS. 1-6, device 10 generally includes ahousing 12 defining a plurality of chambers 14 therein, such as, by wayof example, embodiments of chamber 14 depicted in FIGS. 1-6. Eachchamber 14 includes: a first well region 16 including at least one firstwell 18 and a second well region 20 including at least one second well22. The chamber 14 further includes a channel region 24 including atleast one channel 26 connecting the first well region 16 and the secondwell region 20 with one another. As illustrated in FIGS. 5 and 6, in apreferred embodiment, the first well regions 16 and the second wellregions 20 of the respective ones of the plurality of chambers aredisposed relative to one another to match a pitch of a standardmicrotiter plate. The plurality of chambers may also be disposedrelative to one another to match a pitch of standard microtiter plate.Generally, first well 18 and second well 22 are adapted to receive asample comprising leukocytes and channel 26 is adapted to receiveendothelial cells or leukocyte migration mediators thereon and isconfigured to support physiological shear flow therealong.

In one embodiment of the present invention, channel 26 containsendothelial cells disposed therein. The endothelial cells may beactivated prior to exposure to channel 26 or may have chemokinesimmobilized on the surface opposite the basal lamina therein uponexposure to channel 26. Various cytophilic substances may be disposed inchannel 26 to assist in the attachment of endothelial cells. Cytophilicsubstances are generally substances that have an affinity for cells orsubstances that promote cell attachment to the surface and include, forexample, gelatin, collagen, fibronectin, fibrin, basal lamina,including, but not limited to MATRIGEL™ or other hydrogels.

In another embodiment of the present invention, channel 26 includes aplurality of leukocyte migration mediators disposed therein. Preferably,the plurality of leukocyte migration mediators comprises at least onefirst leukocyte migration mediator and at least one second leukocytemigration mediator, wherein the at least one first and the at least onesecond leukocyte migration mediators are different from one another. Theleukocyte migration mediators are disposed in channel 26 so as to form asurface concentration gradient along a longitudinal axis of chamber 14in increasing concentration from first well 18 to second well 22.

In yet another embodiment of the present invention, channel 26 includeschemokines disposed therein to interact with chemokine receptors on thesurface of rolling leukocytes.

The present invention also contemplates a method of monitoring leukocytemigration. In one embodiment where channel 26 contains endothelial cellsdisposed therein, a sample including leukocytes is placed in first well18 (or second well 22), the sample is allowed to flow along channel 26,the interaction (such interaction including a lack thereof) between theleukocytes and the endothelial cells is observed, and the sampleincluding leukocytes is collected in second well 22 (or the first well18) as the leukocytes exit channel 26. A chemoattractant may be added tochannel 26 to activate the endothelial cells before a sample containingleukocytes is added to first well 18 (or second well 22). In oneembodiment, a test agent is placed in channel 26 and the interactionbetween the leukocytes and endothelial cells in the presence of the testagent is observed.

In one embodiment where channel 26 contains a leukocyte migrationmediator disposed therein, a sample including leukocytes is placed infirst well 18 (or second well 22), the sample is allowed to flow alongchannel 26, the interaction (such interaction including a lack thereof)between the leukocytes and the leukocyte migration mediator is observed,and the sample including leukocytes is collected in second well 22 (orthe first well 18) as the leukocytes exit channel 26. In one embodiment,a test agent is placed in channel 26 and the interaction betweenleukocytes and the leukocyte migration mediator in the presence of thetest agent is observed.

Because device 10, or elements of device 10, may match the footprint ofan industry standard microtiter plate, an advantage of device 10 is thatdevice 10 may be used to conduct multiple assays simultaneously in thesame device, and to high throughput screen various test agents. In onepreferred embodiment, as illustrated in FIG. 5, the first well regions16 and the second well regions 20 of the respective ones of theplurality of chambers 14 are disposed relative to one another to match apitch of a standard microtiter plate. Taking P to designate a pitchbetween respective wells 18/22, the wells may be disposed relative toone another to match a pitch of one of a 24-well microtiter plate, a96-well microtiter plate, a 384-well microtiter plate, 768-wellmicrotiter plate and a 1536-well microtiter plate. By way of example, inthe configuration of chambers 14 as shown in FIG. 5, pitch P will be setto about 9 mm. Preferably, device 10 itself fits in the footprint of anindustry standard microtiter plate. As such, device 10 preferably hasthe same outer dimensions and overall size of an industry standardmicrotiter plate. By way of example, in the configuration of device 10as shown in FIG. 6, device 10 comprises 48 chambers designed in theformat of a standard 96-well plate, such that the respective wells 18/22are disposed relative to one another to match a pitch of a standard96-well microtiter plate with each well fitting in the space of eachwell of the plate. In this embodiment, 48 experiments can be conducted.Alternatively, as seen in FIG. 6A, chambers 14 may be disposed relativeto one another to match a pitch of a standard microtiter plate. In thisalternative embodiment, chambers 14 are sized so that a chamber 14 fitsin the area normally required for a single well of a standard microtiterplate. For example, in this embodiment, device 10, designed in thefootprint of a 96-well microtiter plate configuration, has 96 chambersand therefore allows 96 experiments to be performed. By conforming tothe exact dimensions and specification of standard microtiter plates,embodiments of device 10 would advantageously fit into existinginfrastructures of fluid handling, storage, registration and detection.Device 10 is also conducive to high throughput screening as it allowsrobotic fluid handling and automated detection and data analysis. Theuse of robotic and automated systems also decreases the amount of timeto prepare and perform the assays and analyze the results of the assays.In addition, by using automated systems, the use of device 10 decreasesthe occurrence of human error in preparing and performing assays andanalyzing data. Moreover, because the size of the wells 18/22, or thesize of an entire chamber 12, of device 10 matches the size of a well ofa microtiter plate, the number of leukocytes needed to perform anindividual assay range from only about 103 to about 106. This allows forthe study of rare leukocyte populations, such as basophils or certainlymphocyte subsets. In addition, large amounts of test agents, such asinhibitors and promoters of leukocyte migration, need not be used inorder to conduct assays monitoring the effect of these agents onleukocyte migration.

Based on the preferred configuration of device 10, the present inventionalso contemplates a method of screening a plurality of test agents. Inthis embodiment, the method of screening test agents includes providinga device comprising a housing defining a plurality of chambers. Eachchamber includes: a first well region including at least one first well;a second well region including at least one second well; and a channelregion including at least one channel connecting the first well regionand the second well region with one another. In one embodiment, the atleast one channel includes at least one leukocyte migration mediatordisposed therein. In another embodiment, the at least one channelincludes endothelial cells disposed therein. In both preferredembodiments, at least one of the plurality of chambers on the one hand,and the first well regions and the second well regions of respectiveones of the plurality of chambers on the other hand, are disposedrelative to one another to match a pitch of a standard microtiter plate.The method of screening test agents further includes providingleukocytes in each of the channels of respective ones of the pluralityof chambers; placing at least one of a plurality of test agents in eachof the channels of respective ones of the plurality of chambers; andobserving the interaction between the leukocytes and the endothelialcells or the interaction between the leukocytes and the at least oneleukocyte migration mediator in the presence of the test agents. Forexample, it can be determined whether the test agents have an effect onthe number of leukocytes that are captured, arrested, or havetransmigrated as well as whether the test agents have an effect onvelocity and number of leukocytes that roll along channel 26. The testagent may include any desired biological, chemical, or electricalsubstance, including but not limited to, an inhibitor of leukocytemigration, a promoter of leukocyte migration, or any other therapeuticagent. Further examples of test agents include proteins, nucleic acids,peptides, polypeptides, carbohydrates, lipids, hormones, enzymes, smallmolecules or pharmaceutical agents. This method is particularly usefulin the area of drug discovery where a plurality of test agents may bescreened in a single device 10. Accordingly, it is preferable that eachof the test agents is different from one another and a single test agentis placed in each channel. Of course, if it is desirable to test theeffects of a combination of test agents, for example to determine ifthere is any synergistic effect of two or more test agents, than two ormore test agents of the plurality of test agents may be placed in eachchannel of each of the plurality of chambers.

The device of the present invention may also be used to monitor thesteps of the leukocyte migration cascade under a normal or pathologicalphysiological shear flow condition. A normal physiological flowcondition refers to the shear flow rate during a non-pathological stateand is in the range of about 0.1 dynes/cm² to about 20 dynes/cm². Apathological physiological flow condition refers to the shear flow rateduring the inflammatory response and is generally varied depending onthe disease state. Although the physiological shear flow is preferablyproduced by hydrostatic pressure, or microcapillary action, the flow canbe produced by any means known in the art. For example, if a samplecontaining leukocytes is to be introduced into channel 26 via first well18, then physiological shear flow can be created by applying pressurethrough a vacuum adjacent to second well 22 or by applying pressurethrough a syringe pump adjacent to first well 18. The shear flow may bemanipulated by altering the dimensions of the channels or modifying thedegree of pressure applied through the vacuum or syringe pump.Alternatively, the leukocytes can be introduced into channel 16 in apulsatile manner.

With respect to particular embodiments of device 10 and methods of usingthese embodiments, as mentioned above, channel 26 may have endothelialcells disposed therein or leukocyte migration mediators disposedtherein. The endothelial cells may be disposed on any surface orsurfaces of channel 26. In a preferred embodiment, the endothelial cellsare on the bottom and side surfaces of channel 26. The endothelial cellsmay be disposed uniformly in channel 26, may be disposed in discretepatches, or may be disposed along a concentration gradient such that theconcentration of endothelial cells decreases from first well 18 tosecond well 22. The endothelial cells may be grown on channel 26 in thepresence or absence of shear flow. In one embodiment where channel 26has endothelial cells disposed therein, several different assays may beperformed to observe the interaction between leukocytes and theendothelial cells during the leukocyte migration cascade. For example,to study the process of rolling, a sample containing leukocytes isintroduced into channel 26 via first well 18 or second well 22. Thenumber of leukocytes rolling as well as the rolling velocity of theleukocytes may then be determined. Assays measuring the inhibition ofrolling may also be performed by adding to channel 26, for example,inhibitors that block the interaction between leukocytes and endothelialcells. Similarly, assays measuring the enhancement of rolling may beperformed by adding to channel 26, for example, promoters that promotethe interaction between leukocytes and endothelial cells. A test agentcould also be added to channel 26 to determine the effect of the testagent on the interaction between leukocytes and endothelial cells.

To study the process of arrest, preferably a chemoattractant isintroduced into channel 26 in order to “activate” the endothelium. Thechemoattractants may be any molecule suitable to stimulate theendothelium to express integrin binding ligands such as ICAMs and VCAMs.Non-limiting examples of chemoattractants include cytokines such as IL-1and TNF-α. A sample including leukocytes is then introduced in channel26 via first well 18 or second well 22. Preferably, the sample includingleukocytes is preincubated with a chemoattractant capable of triggeringthe activation of arrest mediator binding partners, for exampleintegrins, on the surface of leukocytes. The chemoattractant is anysuitable substance capable of triggering integrin expression byleukocytes and includes, for example, formyl peptides, intercrines,IL-8, GRO/MGSA, NAP2, ENA-78, MCP-1/MCAF, RANTES, I-309, other peptides,platelet activating factor (PAF), lymphokines, collagen, fibrin, andhistamines. The number of arrested cells may then be determined. Assaysmeasuring the inhibition of arrest may also be performed, for example,by adding inhibitors that block the interaction between chemoattractantsand chemoattractant receptors on the surface of the leukocytes or theendothelium, or that block the interaction between leukocyte arrestmediators and arrest mediator binding partners. A test agent could alsobe added to channel 26 to determine the interaction between theleukocytes and the endothelial cells in the presence of the test agent.

In another embodiment directed to examining the process oftransmigration, a layer of endothelial cells is placed in channel 26. Ina preferred embodiment to more closely simulate in vivo conditions,channel 26 may first be coated with a layer of fibronectin or any otherbasement membrane mimic before adding the endothelial cells to channel26. Preferably the endothelial cells are exposed to eotaxins orchemokines, including RANTES or monocyte chemoattractant protein (MCP-3or MCP-4) prior to introduction of the sample containing leukocytes. Thesample including leukocytes is then introduced into channel 26 via firstwell 18 or second well 22. Preferably, the leukocytes are preincubatedwith a chemoattractant capable of triggering the activation of arrestmediator binding partners, for example integrins, on the surface ofleukocytes. After the leukocytes are allowed to flow along channel 26,the number of cells that transmigrated through the endothelium arecounted. Transmigrated cells may be characterized by appearing flattenedand phase-dark under a microscope. Flattened, phase-dark cells may beconfirmed as being under the endothelial cell monolayer by observing thefocal plane of the leukocytes and the endothelial cells using amicroscope. A cell may be considered transmigrated if, for example,greater than 50% of the cell is under the endothelial cell monolayer atthe point of quantification. Transmigration may be expressed as thenumber of transmigrated cells divided by the total cells counted.Inhibition of transmigration may also be examined by blocking, forexample, the receptor on endothelium cells that binds thechemoattractant responsible for activating the endothelium and thendetermining the number of cells that transmigrate across theendothelium. A test agent may also be introduced in channel 26 todetermine the interaction between the leukocytes and the endothelialcells in the presence of the test agent.

In another embodiment, the endothelial cells disposed in channel 26 havebeen altered or modified through known techniques in molecular biology.For example, the cells may be modified to overexpress particular genesor to not express particular genes coding for the various leukocytemigration mediators responsible for the leukocyte migration cascade.Such an embodiment affords control over the expression of preciseleukocyte migration mediators and allows greater manipulation of themediator involved in the leukocyte migration cascade.

For example, the endothelial cells may be genetically modified to reduceor inhibit the expression of a gene believed to encode a proteininvolved in the leukocyte migration cascade to assist in the elucidationof the proteins involved in leukocyte migration cascade. Methods forgenetically modifying a cell are known in the art. One such method isdisclosed in U.S. Pat. No. 6,025,192 to Beach et al. and involvesreplication-deficient retroviral vectors, libraries comprising suchvectors, retroviral particles produced by such vectors in conjunctionwith retroviral packaging cell lines, integrated provirus sequencesderived from the retroviral particles of the invention and circularizedprovirus sequences which have been excised from the integrated provirussequences of the invention.

In another non-limiting example, the endothelial cells may betransfected with a vector to genetically modify a protein expressed bythe endothelial cells. For various techniques for transfecting mammaliancells, see Keown et al. (1990) Methods in Enzymology 185:527-537. Forexample, the endothelial cells may be modified to express a variant ofthe protein to be tested. For example, if it is believed that a certainprotein is involved in the cascade, the gene expressing the particularprotein can be modified to express a variant. Then using the device andassays of the present invention, the effect of this variant on thevarious parts of the cascade can be monitored.

The variant can be created using techniques known in the art by makingdeletions, additions or substitutions in the sequence encoding theprotein. A “variant” of a polypeptide is defined as an amino acidsequence that is altered by one or more amino acids. Similar minorvariations can also include amino acid deletions or insertions, or both.Guidance in determining which and how many amino acid residues may besubstituted, inserted or deleted without abolishing biological orimmunological activity can be found using computer programs well knownin the art, for example, DNAStar software. A “deletion” is defined as achange in either amino acid or nucleotide sequence in which one or moreamino acid or nucleotide residues, respectively, are absent as comparedto an amino acid sequence or nucleotide sequence of a naturallyoccurring polypeptide. An “insertion” or “addition” is that change in anamino acid or nucleotide sequence which has resulted in the addition ofone or more amino acid or nucleotide residues, respectively, as comparedto an amino acid sequence or nucleotide sequence of a naturallyoccurring polypeptide. A “substitution” results from the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively as compared to an amino acid sequence ornucleotide sequence of a naturally occurring polypeptide. The variantcan have “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant can have “nonconservative”changes wherein a substituted amino acid does not have similarstructural or chemical properties such as replacement of a glycine witha tryptophan.

In addition to creating a variant of the protein of interest, reductionor inhibition of expression of a protein that is expressed by theendothelial cell can be accomplished using known methods of geneticmodification. For example, an endothelial cell expressing a leukocyterolling mediator such as P-selectin can be genetically modified suchthat the expression of the P-selectin is reduced or inhibited using ahomologous recombination gene “knock-out” method (see, for example,Capecchi, Nature, 344:105 (1990) and references cited therein; Koller etal., Science, 248:1227-1230 (1990); Zijlstra et al., Nature, 342:435-438(1989), each of which is incorporated herein by reference; see, also,Sena and Zarling, Nat. Genet., 3:365-372 (1993), which is incorporatedherein by reference). A “knock-out” of a target gene means an alterationin the sequence of the gene that results in a decrease of function ofthe target gene, preferably such that target gene expression isundetectable or insignificant. A knock-out of a gene means that functionof the gene has been substantially decreased so that protein expressionis not detectable or only present at insignificant levels. A “knock-in”of a target gene means an alteration in a host cell genome that resultsin altered expression or increased expression of the target gene, e.g.,by introduction of an additional copy of the target gene, or byoperatively inserting a regulatory sequence that provides for enhancedexpression of an endogenous copy of the target gene.

The expression of the leukocyte migration mediator by an endothelialcell also can be reduced or inhibited by providing in the endothelialcell an antisense nucleic acid sequence, which is complementary to anucleic acid sequence or a portion of a nucleic acid sequence encoding aleukocyte migration mediator. Methods for using an antisense nucleicacid sequence to inhibit the expression of a nucleic acid sequence areknown in the art and described, for example, by Godson et al., J. Biol.Chem., 268:11946-11950 (1993), which is incorporated herein byreference.

Another embodiment creating control over the precise leukocyte migrationmediators to be studied, including control over the type and amount ofleukocyte migration mediator expressed, involves an embodiment of device10 wherein at least one leukocyte migration mediator is disposed inchannel 26 therein. For the purpose of clarity, the term “leukocytemigration mediator” used herein necessarily refers to at least oneleukocyte migration mediator, unless otherwise specified. By disposing aleukocyte migration mediator in channel 26, it is possible to examinethe ligand/receptor interactions underlying the leukocyte migrationcascade, including the individual events of the cascade to gain afurther understanding of this process. Disposing a leukocyte migrationmediator in channel 26 also allows for the precise targeting of theligand/receptor interactions underlying the individual events of theleukocyte migration cascade.

For example, in one embodiment, device 10 may be used to examine thecapture of a leukocyte wherein the leukocyte migration mediator disposedin channel 26 may comprise a leukocyte capture mediator. As aconsequence of the initial immune response to infection, inflammatorymediators induce the expression of adhesion molecules on the surface ofthe endothelium, resulting in an “activated endothelium.” The firstcontact of a leukocyte with the activated endothelium is known as“capture” and is thought to involve a capture mediator P-selectin and acapture mediator binding partner L-selectin. P-selectin is thought to bethe primary adhesion molecule involved the capture process and thebinding of P-selectin to its main capture mediator binding partner,PSGL-1, is strongly implicated in this process. L-selectin has also beenimplicated in capture although its precise ligand on endothelial cellsis unknown. Accordingly, in this embodiment of the present invention,the leukocyte capture mediator disposed in channel 26 may comprise, forexample, P-selectin and/or an L-selectin binding ligand.

In another embodiment of the present invention, device 10 may be used toexamine the rolling of a leukocyte wherein the leukocyte migrationmediator may comprise a leukocyte rolling mediator. Once leukocytes arecaptured they may transiently adhere to the endothelium and begin toroll along the endothelium. The rolling of leukocytes is thought toinvolve: a rolling mediator, P-selectin; a rolling mediator bindingpartner, L-selectin; and a rolling mediator, E-selectin, althoughP-selectin is considered the primary adhesion molecule involved in thisprocess. Accordingly, in this embodiment of the present invention, theleukocyte rolling mediator disposed in channel 26 may include, forexample P-selectin, E-selectin, and/or an L-selectin ligand.

In another embodiment of the present invention, device 10 may be used toexamine the arrest of a leukocyte wherein the leukocyte migrationmediator may comprise a leukocyte arrest mediator. It is thought thatmost, if not all, leukocytes adhere to the endothelium only after havingrolled along the endothelium. This adhesion, or “arrest” of theleukocytes on the top surface of the endothelium is initiated bychemoattractants such as IL-1 and TNF-α produced by cells at the injuredsite. These chemoattractants stimulate the endothelium to producechemokines and arrest mediators on the surface of the endotheliumopposite the basal lamina. The arrest mediators comprise, for example,integrin binding ligands such as ICAMs, including ICAM-1, ICAM-2, or/andICAM-3 and VCAMs, including VCAM-1 and/or VCAM-2. The chemokinesinteract with chemokine receptors on the surface of the rollingleukocytes, which triggers the activation of arrest mediator bindingpartners on the surface of leukocytes. Arrest mediator binding partnersinclude integrins, such as, for example, LFA-1, Mac-1, and p150,95, andVLA-4. Activation of these arrest mediator binding partners is thoughtto cause the slowly rolling leukocytes to “arrest” and strongly bind tothe arrest mediators, such as ICAM-1, VCAM-1, and other integrin bindingligands such as collagen, fibronectin, and fibrinogen, on theendothelium. Accordingly, in this embodiment of the present invention,the leukocyte arrest mediator disposed in channel 26 may include atleast one integrin binding ligand.

In yet another embodiment of the present invention, device 10 may beused to examine the transmigration of a leukocyte wherein the leukocytemigration mediator disposed in channel 26 comprises a leukocytetransmigration mediator. Once bound to the endothelium, the leukocytesflatten and squeeze between the endothelium to leave the blood vesseland enter the damaged tissue. The leukocytes follow a chemotacticgradient of chemoattractants released by cells in the damaged tissuearea. Although much still remains unknown about transmigration,transmigration is thought to be mediated by platelets and endothelialcell adhesion molecule-1 (PECAM-1). Other potential transmigrationmediators may be junctional adhesion molecule (JAM), ICAM-,1VE-cadherin, LFA-1, IAP, VLA-4 and possibly CD99, a transmembraneprotein. Accordingly, in this embodiment of the present invention, theleukocyte transmigration mediator disposed in channel 26 may include atleast one of the aforementioned adhesion molecules or any other moleculedetermined to be implicated in transmigration.

Device 10 of the present invention may be used to study eachaforementioned step in the leukocyte migration cascade in isolation, acombination of two or more steps in the leukocyte migration cascade, orthe leukocyte migration cascade in its entirety. For example, ifunderstanding and targeting rolling are desired, then preferably onlyleukocyte rolling mediators may be disposed in channel 26. If bothrolling and arrest of leukocytes are desired to be studied, then bothrolling and arrest mediators may be disposed in channel 26. If theentire leukocyte migration cascade is to be examined, then capturemediators, rolling mediators, arrest mediators, and transmigrationmediators may be disposed in channel 26. It is understood that becausecertain molecules belong in more than one category of migrationmediators (for example P selectin and an endothelial ligand bindingL-selectin function as both capture mediators and rolling mediators) andbecause certain mediators may be present in conjunction (for example tostudy arrest, both rolling and arrest mediators may be present inchannel 26 since direct adhesion from free-flowing leukocytes is thoughtto be extremely rare), certain steps, with the knowledge currentlyavailable, may not be monitored in isolation. Because much is stillunknown about the specific details of the cascade of events occurringduring the inflammatory response, this invention contemplates severalmethods of monitoring leukocyte migration in order to gain furtherunderstanding of the basic mechanisms controlling these events.

For example, to study the process of capture, a leukocyte migrationmediator comprising a capture mediator is disposed in channel 26. Asample comprising leukocytes is introduced into channel 26 via firstwell 18 or second well 22. Capture events are defined as adhesiveinteractions of those freely flowing leukocytes moving closest to thesurface of channel 26 containing the capture mediators and that aretherefore the only leukocytes potentially capable of interacting withthe capture mediators on channel 26. Different types of initialleukocyte capture can be characterized, observed, and monitored. Forexample, transient capture involving leukocytes only attaching brieflyto channel 26 without initiating rolling motions, and rolling captureinvolving leukocytes that remain rolling on channel 26, can bedetermined. The number of each type of captured leukocyte can be dividedby the total number of free flowing leukocytes to determine thefrequency of initial capture of leukocyte.

The leukocytes can also be observed via any method known in the art andvia methods disclosed in co-pending application entitled “Test Deviceand Method of Making Same,” which is herein incorporated by reference inits entirety. Briefly, the leukocytes may be observed by using amicroscope, including phase-contrast, fluorescence, luminescence,differential-interference contrast, dark field, confocal laser-scanning,digital deconvolution, and video microscopes; a high-speed video camera;and an array of individual sensors. For example, a digital movie cameramay be used to monitor leukocyte activity under continuous flowconditions or a camera may be used to obtain still photographic imagesat particular points in time. Such observations reveal the interactionbetween the capture mediator binding partner expressed by the leukocytesand the capture mediator expressed by the endothelium. To detect suchinteraction, the cells may be incubated with staining agents and thendetected based upon color or intensity contrast using any suitablemicroscopy technique(s). Alternatively, fluorescence-labeling may beused to detect whether capture mediator binding partners bind to capturemediators.

In another embodiment, non-labeled cells may be used to monitormigration. For example, a heterogeneous mixture of multiple cell typesmay be introduced into channel 26 with only one cell type capable ofinteracting with the capture mediators in channel 26. After the cellshave been introduced into channel 26, an antibody specific to anyantigen on the surface of this cell type may be used to label this celltype. If a multiple number of cell types can interact with the capturemediators, antibodies labeled with specific fluorophores can be used todistinguish different cell types.

In another embodiment directed to examining the process of rolling, aleukocyte migration mediator comprising a rolling mediator is placed inchannel 26. A sample comprising leukocytes is introduced into channel 26via first well 18 or second well 22. The number of leukocytes rollingand the rolling velocity of the leukocytes can be determined. In oneembodiment, a camera is operatively linked to device 10 to obtain imagesof leukocytes rolling along channel 26 during predetermined intervalsover a predetermined period of time. In this embodiment, the rollingvelocity of the cells is determined by measuring the length the cellstraveled (1_(frame)) in an image obtained by the camera and determiningthe exposure time of the image (t_(exposure)). To determine the rollingvelocity (V), the following formula is used:V=c(1_(frame) /t _(exposure))where c is a conversion factor for determining the actual distance thecells have traveled. It may vary from image to image.

In another embodiment, several different assays utilizing differenttypes of leukocytes are performed to characterize and compare therolling velocities associated with the different cell types. In anotherembodiment, several different assays utilizing different rollingmediators are performed to characterize and compare the rollingvelocites of cells associated with the different rolling mediators.

In another embodiment directed to examining the process of arrest, aleukocyte migration mediator comprising a first leukocyte migrationmediator and a second leukocyte migration mediator, the first and secondleukocyte migration mediators being different from one another isutilized. For the purpose of clarity, the term “first leukocytemigration mediator” used herein necessarily refers to at least one firstleukocyte migration mediator and the term “second leukocyte migrationmediator” used herein necessarily refers to at least one secondleukocyte migration mediator. In this embodiment, the first leukocytemigration mediator comprising a rolling mediator and the secondleukocyte migration mediator comprising an arrest mediator are placed inchannel 26. A fluid sample comprising leukocytes is preincubated with achemoattractant capable of triggering the activation of arrest mediatorbinding partners, for example, integrins, on the surface of theleukocytes. The chemoattractant is any suitable substance capable oftriggering integrin expression by leukocytes and includes, for example,a formyl peptide, intercrines, IL-8 GRO/MGSA,NAP-2, ENA-78, MCP-1/MCAF,RANTES, I-309, other peptides, platelet activating factor (PAF),lymphokines, collagen, fibrin and histamines. The number of arrestedcells can then be determined and assays similar to those performed withonly rolling mediators can be performed.

In order to further understand the biological influences that underliethe leukocyte migration cascade, particularly the interaction betweenleukocytes and their counter-receptors on the endothelium, device 10 mayalso be used to analyze the effects of various test agents on theleukocyte migration cascade. These test agents may comprise anybiological, chemical or electrical substance that includes, but is notlimited to potential inhibitors of the leukocyte migration mediator orpotential promoters of migration mediated by the leukocyte migrationmediator. Further examples of such test agents include proteins,peptides, polypeptides, enzymes, hormones, lipids, carbohydrates, smallmolecules, and pharmaceutical agents. For example, the device may beused to identify an inhibitor or promoter that competitively ornoncompetitively inhibits or promotes a capture mediator and capturemediator binding partner interaction; rolling mediator and rollingmediator binding partner interaction; arrest mediator and arrestmediator binding partner interaction; and/or transmigration mediator andtransmigration mediator binding partner interaction. As mentionedearlier, preferably the leukocyte migration mediator comprises a firstleukocyte migration mediator and a second leukocyte migration mediator,the first and second leukocyte migration mediators beings different fromone another. As such, in one embodiment, the test agent comprises apotential inhibitor of the first leukocyte migration mediator, thesecond leukocyte migration mediator, or both. In another embodiment, thetest agent comprises a potential promoter of migration mediated by thefirst leukocyte migration mediator, the second leukocyte migrationmediator, or both. After identifying inhibitors and promoters of theleukocyte migration cascade, these inhibitors and promoters can betested for efficacy in vivo and ultimately utilized as therapeuticagents.

To screen a test agent that is a potential inhibitor of capture, aleukocyte migration mediator comprising a capture mediator is disposedin channel 26. After the potentially inhibitory test agent is incubatedwith a fluid sample comprising leukocytes, the sample is introduced intochannel 26 via first well 18 or second well 22. Capture events aredefined as adhesive interactions of those freely flowing leukocytesmoving closest to the surface of channel 26 containing the capturemediators and that are, therefore, the only leukocytes potentiallycapable of interacting with the capture mediators on channel 26.Different types of initial leukocyte capture can be characterized,observed, and monitored. For example, transient capture involvingleukocytes only attaching briefly to channel 26 without initiatingrolling motions, and rolling capture involving leukocytes that remainrolling on channel 26 can be determined. The number of each type ofcaptured leukocyte can be divided by the total number of free flowingleukocytes to determine the frequency of initial capture of leukocytesincubated with the potential inhibitory test agent and this frequencycan be compared to the frequency of initial leukocyte capture in theabsence of the potential inhibitory test agent. If the frequency ofinitial leukocyte capture is lower in the presence of the test agentrelative to the frequency of initial leukocyte capture in the absence ofthe test agent, then the test agent is likely an inhibitor of leukocytecapture.

To screen a test agent that is a potential inhibitor of rolling, aleukocyte migration mediator comprising a rolling mediator is placed inchannel 26. After the potentially inhibitory test agent is incubatedwith a fluid sample comprising leukocytes, the sample is introduced intochannel 26 via first well 18 or second well 22. Alternatively, thepotentially inhibitory test agent is introduced into the fluid sampleduring passage of the fluid sample in channel 26 when leukocytes havebegun rolling. A decrease in rolling (e.g. as measured by a decrease intheir velocity, or a decrease in the number of rolling leukocytes pervolume) in the presence of the test agent, relative to that observed inthe absence of the test agent, may indicate that the molecule is aninhibitor of capture and/or rolling.

To test a potentially inhibitory test agent of arrest, a leukocytemigration mediator comprising a first leukocyte migration mediator and asecond leukocyte migration mediator, the first and second leukocytemigration mediators being different from one another may be utilized. Inthis embodiment, the first leukocyte migration mediator comprising arolling mediator and the second leukocyte migration mediator comprisingan arrest mediator are placed in channel 26. A fluid sample comprisingleukocytes is preincubated with a chemoattractant capable of triggeringthe activation of arrest mediator binding partners, for example,integrins, on the surface of the leukocytes. After the fluid sample ispreincubated with the potentially inhibitory test agent, the sample isintroduced into channel 26 via first well 18 or second well 22.Alternatively, the potentially inhibitory test agent is introduced intothe fluid sample during passage of the fluid sample in channel 26 whenleukocytes have begun rolling. A decrease in arrest of the leukocytes(e.g., as measured by a decrease in the percentage of leukocytes thatare arrested, or in the number of arrested leukocytes per volume) in thepresence of the test agent relative to that observed in the absence ofthe test agent, indicates that the test agent may be an inhibitor ofleukocyte arrest.

Device 10 can also be used to identify whether a test agent acts as apromoter of the inflammatory response by increasing the efficiency ofthe leukocyte migration cascade or by acting as a functional componentthereof (e.g. a capture mediator, a rolling mediator, an arrestmediator, or a transmigration mediator). Such a functional component maybe detected by its ability to promote capture, rolling, arrest ortransmigration of a leukocyte where such action was previously lacking(due to lack of appropriate cellular specificity of a rolling mediatoror arrest mediator previously present in channel 26 of chamber 14 orlack of any rolling mediator or arrest mediator). For example, device 10comprising a first leukocyte migration mediator comprising a rollingmediator and second leukocyte migration mediator comprising an arrestmediator disposed in channel 26 may be used to identify an arrestmediator functional in leukocyte migration. In addition, device 10comprising an arrest mediator and/or a rolling mediator disposed inchannel 26 may be used to identify a rolling mediator functional inleukocyte migration.

To identify an arrest mediator, rolling mediators are disposed inchannel 26 that have rolling binding partners present on the surface ofleukocytes in a fluid sample to be introduced into channel 26 throughfirst well 18 or second well 22. One or more chemoattractants capable ofactivating the leukocytes to express arrest mediator binding partnersare preincubated with the fluid sample comprising leukocytes. A testagent to be tested for arrest mediating function is disposed in channel26. After the fluid sample comprising leukocytes is introduced intochannel 26 via first well 18 or second well 22 and the sample passesalong channel 26, it is determined whether any leukocytes have arrestedon channel 26. Arrest of leukocytes indicates that the test agent may bean arrest mediator that recognizes an arrest mediator binding partner onthe surface of the same leukocytes that express the rolling mediatorbinding partner.

To identify a rolling mediator, the test agent to be tested for rollingmediator function is disposed in channel 26 and the fluid samplecomprising leukocytes is introduced into channel 26 via the first well18 or second well 22 and the sample is allowed to flow along channel 26.Rolling of the leukocytes along channel 26 indicates that the test agenthas rolling mediator function and that the leukocytes express a bindingpartner for the rolling mediator. A rolling mediator is also identifiedby disposing the test agent to be tested for rolling mediator functionin channel 26 and also disposing an arrest mediator in channel 26. Oneor more chemoattractants capable of activating the leukocytes to expressarrest mediator binding partners, such as integrins, are preincubatedwith the fluid sample comprising leukocytes. The fluid sample comprisingleukocytes is then introduced into channel 26 via the first well 18 orsecond well 22 and the sample is allowed to flow along channel 26.Arrest of leukocytes indicates that the test agent has rolling mediatorfunction and that the leukocytes that express the arrest mediatorbinding partner and the chemoattractant receptor also express a bindingpartner for the test agent.

A test agent may also be identified as a functional component in theprocesses of leukocyte rolling or rolling and arrest, or an enhancerthereof, by the aforementioned methods in which an increase in number orpercentage of leukocytes rolling or arrested is detected relative to thenumber or percentage of such leukocytes in the absence of the testagent. The migration of the leukocytes may be observed, monitored,recorded, and analyzed by any method known in the art and via themethods disclosed in co-pending application, “Test Device and Method ofMaking Same” referred to above.

The present invention also provides a kit to conduct the aforementionedassays. For example, the kit comprises a device including a housing 12defining a plurality of chambers 14. Each of the plurality of chambers14 includes a first well region 16 including at least one first well 18;a second well region 20 including at least one second well 22; and achannel region 24 including at least one channel 26 connecting the firstwell region 16 and the second well region 20 with another. The firstwell regions 16 and the second well regions 20 of the respective ones ofthe plurality of chambers 14 are preferably disposed relative to oneanother to match a pitch of a standard microtiter plate, thusadvantageously allowing for high through-put screening of tests agents.The kit further includes a first leukocyte migration mediator. The kitmay also contain a sample comprising leukocytes. The first leukocytemigration mediator and the sample comprising leukocytes may be packagedin the kit in any manner known in the art. For example, the firstleukocyte migration mediator may be contained in a vial or container andthe sample comprising leukocytes may similarly be contained in aseparate vial or container. The kit may further include a secondleukocyte migration mediator different from the first leukocytemigration mediator. The kit may additionally include an inhibitoradapted to inhibit the first leukocyte migration mediator, the secondleukocyte migration mediator, or both. The kit may further include apromoter adapted to promote migration mediated by the first leukocytemigration mediator, the second leukocyte migration mediator, or both.The kit may also include any media and buffers necessary for use withthe device and particular assays.

With respect to particular details of device 10, preferably, as shown byway of example in FIGS. 1 and 6, the housing 12 of device 10 comprises asupport member 28, and a top member 30 mounted to the support member 28,wherein the support member 28 and the top member 30 are configured suchthat they together define the plurality of chambers 14. Preferably, thehousing is also sized to match dimensions of a standard microtiterplate, for example, the dimensions of a 24-well microtiter plate, a96-well microtiter plate, a 384-well microtiter plate, 768-wellmicrotiter plate and a 1536-well microtiter plate. The top member may bemade of any suitable material known in the art including glass, plastic,or an elastomeric material such as polydimethylsiloxane (PDMS). Thesupport member may be made of glass, polystyrene, polycarbonate,polyacrylates, polymethyl methacrylate (PMMA), PDMS and other plastics.Preferably, top member 30 is in conformal contact with support member28.

In another embodiment of the present invention, device 10 comprises asupport member 28; and top member 30, the top member 30 mounted to thesupport member 28 by being placed in conformal contact with the supportmember 28. The support member 28 and the top member 30 are configuredsuch that they together define at least one chamber 14. The at least onechamber 14 includes a first well region 16 including at least one firstwell 18; a second well region 20 including at least one second well 22;and a channel region 24 including at least one channel 26 connecting thefirst well region 16 and the second well region 20 with one another. Inone embodiment, the at least one channel 26 includes at least oneleukocyte migration mediator disposed therein. In another embodiment,the at least one channel 26 includes endothelial cells disposed therein.Preferably, the top member 30 is configured to be placed in reversible,conformal contact with the support member 28. As such, top member 30 ispreferably made of a material that is adapted to effect conformalcontact, preferably reversible conformal contact, with support member28. According to this embodiment, the flexibility of such a material,among other things, allows top member 30 to form-fittingly adhere tosupport member 28 in such a way as to form a substantially fluid-tightseal therewith. The conformal contact should preferably be strong enoughto prevent slippage of top member 30 on support member 28. Where theconformal contact is reversible, top member 30 may be made of a materialhaving the structural integrity to allow top member 30 to be removed bya simple peeling process. This would allow top member 30 to be removedfrom support member 28 after experimentation, properly cleansed, andthen reused for future assays. Preferably, the peeling process does notdisturb any surface treatment, such as leukocyte migration mediators orendothelial cells, on support member 28. Additionally, the substantiallyfluid-tight seal effected between top member 30 and support member 28 byvirtue of the conformal contact of top member 30 with support member 28prevents fluid from leaking from one chamber to an adjacent chamber, andalso prevents contaminants from entering the wells. The seal preferablyoccurs essentially instantaneously without the necessity to maintainexternal pressure. The conformal contact obviates the need to use asealing agent to seal top member 30 to support member 28. Althoughembodiments of the present invention encompass use of a sealing agent,the fact that such a use is obviated according to a preferred embodimentprovides a cost-saving, time-saving alternative, and further eliminatesa risk of contamination of each chamber 14 by a sealing agent.Preferably, the top member 30 is made of a material that does notdegrade and is not easily damaged by virtue of being used in multipletests, and that affords considerable variability in the top member'sconfiguration during manufacture of the same. More preferably, thematerial may be selected for allowing the top member 30 to be made usingphotolithography. In a preferred embodiment, the material comprises anelastomer such as silicone, natural or synthetic rubber, orpolyurethane. In a more preferred embodiment, the material is PDMS.Support member 28 provides a support upon which top member 30 rests, andmay be made of any material suitable for this function. Suitablematerials are known in the art such as glass, polystyrene,polycarbonate, PMMA, polyacrylates, PDMS, and other plastics.

With respect to portions of chamber 14, in one embodiment, well regions16 and 20 are vertically offset with respect to one another is a testorientation of device 10. In a preferred embodiment, well regions 16 and20 are horizontally offset with respect to one another is a testorientation of device 10. Wells 18 and 22 of respective well regions 16and 20 of each chamber 14 are not limited in their configuration to anyparticular three dimensional contour, it being only required that theybe adapted to receive a fluid therein, preferably a sample comprisingleukocytes. Preferably, wells 18 and 22 are configured such that theysubstantially define circles in top plan views thereof, as shown by wayof example in FIGS. 1-6. However, other contours in the top plan view ofa given well is within the scope of the present invention, as readilyrecognized by one skilled in the art. Where the wells define circles intop plan views thereof, and where, the well regions are disposedrelative to one another to match a pitch of a standard 96-wellmicrotiter plate, the pitch P is set to be equal to about 9 mm, and thediameter D_(w) of a top plan contour of the wells is set to be equal toabout 6 mm. In such a case, length L of each channel 26 is equal toabout 3 mm. As shown in particular in FIG. 2, wells 18 and 22 aredefined in part by respective through-holes 18 ¹ and 22 ¹ in top member30, and in part by an upper surface U of support member 28. Inparticular, the sides of each well 18 and 22 are defined by respectivewalls of the through holes 18 ¹ and 22 ¹ in the top member 30, and thebottoms of wells 18 and 22 are defined by a corresponding portion of theupper surface U of support member 28.

With respect to channel region 24 of chamber 12, as seen collectively inFIGS. 2-4, a length L of a channel 26 is defined in a direction of thelongitudinal axis thereof. In addition, depth D of a channel 26 isdefined in a direction normal to a top surface of housing 12; and awidth W of a channel region 26 is defined in a direction normal tolength L and depth D. Preferably, channel region 24 comprises aplurality of rectilinear, parallel channels 26 extending between wellregions 16 and 20. Preferably, channels 26 have lengths L that aresubstantially identical, as shown schematically by way of example inFIG. 4. More preferably, the plurality of channels 26 comprises eightchannels. By using multiple channels, multiple assays can be performedsimultaneously using one sample comprising leukocytes. In such anembodiment, all assays are performed under uniform and consistentconditions and therefore provide statistically more accurate results.Channels 26 preferably each have a width W of 50 μm to 5 mm; a length Lof about 1-10 mm; and a height H of about 10-100 μm. More preferably,the channels 26 each have a width W of about 100 microns, a length L ofabout 3 mm and a height of about 50 μm to about 80 μm. The dimensions ofthe channels 26 of channel region 24 should be configured to support themigration of leukocytes under conditions simulating such migrationduring an inflammatory response. As such, the channel region should beadapted to support the migration of leukocytes under shear flow and tosupport at least one leukocyte migration mediator disposed therein. Itis to be noted that the embodiments of device 10 described in relationto FIGS. 1-6 are merely exemplary, and that various other configurationsare within the scope of the present invention. Other examples for theconfiguration of device 10 are provided in the co-pending applicationentitled “Test Device and Method of Making Same,” referenced to above.

Device 10 of the present invention can be fabricated, according to apreferred embodiment of a method of the present invention, by standardphotolithographic procedures. Photolithographic procedures can be usedto produce a master that is the negative image of any desiredconfiguration of top member 30. For example, the dimensions of chamber14, such as the size of well region 16 and 18, or the length of channelregion 24, can be altered to fit any advantageous specification. Once asuitable design for the master is chosen and the master is fabricatedaccording to such a design, the material for top member 30 is eitherspin cast, injected, or poured over the master and cured. Once the moldis created, this process can be repeated as often as necessary. Thisprocess not only provides great flexibility in the top member's design,it also allows the top members to be massively replicated.

Once the device is fabricated, leukocyte migration mediators can bedisposed in channel 26. The leukocyte migration mediators can bedisposed in channel 26 by affixing them or physioadsorbing them directlyon the upper surface U of support member 28, or by coating a solution orsuspension comprising the leukocyte migration mediators on the uppersurface U of support member 28, as long as the mediators are accessibleto leukocytes flowing by the upper surface U. In one embodiment, theleukocyte migration mediators are either covalently or non-covalentlyaffixed directly to upper surface U by techniques such as covalentbonding via an amide, ester or lysine side chain linkage or adsorption.Other method of disposing leukocyte migration mediators, includingimmobilizing them on upper surface U of support member 28 are disclosedin co-pending application, “Test Device and Method of Making Same”referred to above.

The present invention also provides a device comprising a housing 12;means associated with the housing defining a plurality of chambers 14 inthe housing 12. Each of the plurality of chambers 14 includes: an inletmeans for receiving a sample comprising leukocytes; an outlet means inflow communication with the inlet means for receiving the samplecomprising leukocytes from the inlet means; and connection meansconnecting the inlet means and the outlet means to one another, theconnection means including at least one leukocyte migration mediatordisposed therein. An example of means associated with the housingdefining a plurality of chambers in the housing comprises a top membermounted to a support member as shown in FIG. 6. The above means havebeen substantially shown and described in relation to the embodiments ofthe FIGS. 1-6. Other such means would be within the knowledge of personsskilled in the art.

In another embodiment, the present invention provides methods ofassaying and studying biological phenomenon that either depend on orreact to gradient formation and/or flow conditions. Such biologicalphenomenon include many of the processes in the body such ascell-surface interactions such as that occurring during leukocyteadhesion and rolling. In addition, studies involving chemotaxis,haptotaxis and cell migration will be better served with assays that areable to study such cell movement in the presence of gradients and/orflow conditions.

Various types of gradients are useful in the study of biologicalsystems. Such useful gradients include static gradients, which haveconcentrations that are fixed, or set or substantially fixed or set. Oneexample of a static gradient is a gradients of immobilized molecules ona surface. Non-limiting examples of static gradients include the use ofdiffering concentrations of immobilized biomolecules (proteins,antibodies, nucleic acids, and the like) or immobilized chemicalmoieties (drugs and small molecules). Other useful gradients includedynamic gradients, which have concentrations that may be varied. Oneexample of a dynamic gradient is a gradient of fluid streams havingmolecules in varying concentrations. Non-limiting examples of fluidgradients include the use of fluid streams containing biomolecules suchas growth factors, toxins, enzymes, proteins, antibodies, carbohydrates,drugs or other chemical and small molecules in varying concentrations.

In one embodiment of the present invention, a dynamic/solution basedgradient is created by laminar flow technology. Laminar flow technologytypically involves two or more fluid streams from two or more differentsources. These fluid streams are brought together into a single streamand are made to flow parallel to each other without turbulent mixing.Fluids with different characteristics such as varying low Reynoldsnumbers will flow side by side and will not mix in the absence ofturbulence. Since the fluids do not mix, they create pseudo-channels(pseudo by the fact that there are no physical separation between thefluids). The generation of solution and surface gradients is discussedin U.S. patent application 2002/0113095 and an article, Jeon, Noo Li, etal., Langmuir, 16, 8311-8316 (2000). Both of these references are hereinincorporated by reference in their entirety.

In these references a PDMS microfluidic device was used to generate agradient through a microfluidic network of capillaries. Solutionscontaining different chemicals were introduced into three separateinlets and allowed to flow through the network of capillaries. The fluidstreams were repeatedly combined, mixed, and split to yield distinctmixtures with distinct compositions in each of the branching channels.As illustrated in FIG. 13, when all of the branches were recombined, aconcentration gradient was established across the outlet channel,perpendicular to the flow direction.

By combining the devices of the present invention with the formation ofa dynamic gradient, a vast number of assay parameters can be generatedby altering any portion of the device. For example, by combining thedevice as disclosed herein with cell patterning techniques, along withthe introduction of a dynamic gradient, various conditions can becreated to test numerous biological interactions. Further, the deviceand assays may be useful in drug discovery and drug testing as manycells and biological materials behave differently ex vivo when notexposed to gradients than compared to when the cells or biologicalmaterials are present in vivo and thus exposed to gradients and flowconditions.

Accordingly, in one embodiment of the present invention, cells can bepatterned across channel 26. Cell patterning can be achieved by methodsknown in the art, as well as disclosed in the present invention (suchas, but not limited to, microcontact printing or by the use ofelastomeric stencils). A solution containing any desired biomolecule orchemical/drug can then be flowed across the patterned cells.Additionally, the cells could be first treated by a biomolecule such asan activator to more closely recreate a biological system, and then besubsequently exposed to a chemical or drug. By creating a gradient, suchas by laminar flow, different amounts of biomolecules or chemicals/drugscan be delivered to the patterned cells and thus the effect ofconcentration of each biomolecule or chemical/drug be testedsimultaneously against each other. This side by side, same timecomparison thus reduces the variability of assay to assay conditions.

Creating dynamic gradients with laminar flow in combination with thedevices of the present invention provides numerous assay configurations.For example, by varying the combinations of the cells on the surface,the biomolecule in channels 26 and the compounds in channel 26, one cancreate a vast multitude of assays.

With respect to immobilized cells or other immobilized biomolecules suchas proteins, antibodies, nucleic acids, etc. different assayconfigurations are possible. In one embodiment, a single type of cell isimmobilized throughout the entire channel region 24. In anotherembodiment, a mixture of cell types are immobilized, one cell type perregion 24. In another embodiment, a mixture of cell types is immobilizedthroughout the entire channel region 24. This may be advantageous inmonitoring cell-cell interactions. In yet another embodiment, differentcell types are immobilized in each different region 24.

In addition to the various immobilization schemes, further assay designflexibility centers around the leukocyte migration mediators or otherbiomolecules present in channels 26. For example, in one embodiment, onetype of leukocyte migration mediator is present in each channel 26 atthe same concentration. In another embodiment, one type of leukocytemigration mediator is present in each channel 26 at differingconcentrations. In another embodiment, different leukocyte migrationmediators are present in each channel 26. In another embodiment, thereis a mixture of leukocyte migration mediators in each channel 26. Eachchannel 26 may have the same mixture or a different mixture. When themixture is the same, the ratios or concentrations of the differentleukocyte migration mediators may be different in each channel 26.

Likewise with respect to compounds, such as drugs or test substances,the present invention provides flexibility in assay design. For example,in one embodiment a single compound is present in all channels 26 at thesame concentration throughout. In another embodiment, the same compoundis present in all the channels 26 but each channel 26 has a differentconcentration of that compound. In another embodiment, each channel 26has a different compound. In another embodiment, there is more than onecompound. When there is more than one compound, each channel 26 may havethe same mixture of compounds or may have a different mixture ofcompounds. Further, when the mixtures of the compounds are the same,each channel 26 may receive a different concentration of that mixture.Yet, even further, each channel 26 may receive the mixture of thecompounds, with each channel 26 having a different ratio of compounds toeach other.

Such assay systems can be used to test among many numerous biologicalinteractions, the effects of chemical or drugs on cells or otherbiomolecules. For example, one may use the device and the assays of thepresent invention to measure the IC50 of a compound by using a laminarflow gradient of a compound present from a low concentration to a highconcentration flowed across immobilized biomolecules.

Throughout this application, reference has been made to variouspublications, patents, and patent applications. The teachings anddisclosures of these publications, patents, and patent applications intheir entireties are hereby incorporated by reference into thisapplication to more fully describe the state of the art to which thepresent invention pertains.

EXAMPLES

I. Procedure for Fabrication of the Device for Monitoring LeukocyteMigration

A master of the device according to the present invention is made usingphotolithography. A silicon substrate is patterned based on a negativepattern of the top member using a suitable photoresist. Thereafter,polydimethyl siloxane (PDMS) is poured on top of the master and placedunder vacuum in order to extract air bubbles therefrom. The thus pouredPDMS layer is allowed to cure in an oven at about 30° C. for about 17hours. Thereafter, the device is washed thoroughly with 2% Micro-90 (aproduct of International Products Corp.), rinsed for 10 minutes at 70°C. in “Sonic Bath,” and rinsed with de-ionized water, followed by arinsing with 100% ethanol. The PDMS layer is then dried under nitrogen.At the same time, a pre-cleansed glass slide, such as a rectangular onehaving dimensions of about 4.913+/−0.004 inches (in.) by about3.247+/−0.004 in. and a thickness of about 1.75 millimeters (mm), mm, iswashed three times with ethanol and twice with methanol. Preferably, thesurfaces of the PDMS layer and the glass slide to be bound together areboth plasma oxidized for about 84 seconds. The PDMS layer and the glassslide are then pressed together using forceps to squeeze out air pocketstherebetween. In this manner, a fluid-tight, conformal contact isestablished between the PDMS layer as top member and the glass slide assupport member. In addition, by virtue of PDMS having been used as thetop member material, the conformal contact between the PDMS layer andthe glass slide is reversible.

It is to be noted that the method of making the device of the presentinvention described above is merely an example. Other examples for themethod of making the device are provided in the co-pending applicationentitled “Test Device and Method of Making Same” referred to above.

II. Leukocyte Migration Assay Utilizing Device of the Present Inventionwith a Rolling Mediator Disposed in a Channel Therein

A. Isolation of Leukocytes

Neutrophils are isolated from a volume of 5 milliliters (ml) of humanblood from a healthy volunteer. The 5 ml of blood is diluted with HanksBalance Salt Solution (HBSS) in a 1:2 ratio thereby increasing the totalvolume of blood to equal 15 ml. The whole blood dilution is layered over10 ml of Ficoll-Paque Plus (obtained from Amersham Pharmacia Biotech AB,catalog # 17-1440-02). The blood is then centrifuged for 30 minutes at400 g at room temperature. The supernatant is aspirated off withoutdisturbing the pellet. The pellet is resuspended on 10 ml of HBSS and150 μl of 6% dextran to make up a 1% solution. The red blood cells areallowed to settle for at least one hour at room temperature. Theneutrophils remain inside the supernatant while the red blood cellsmostly settle down forming a pellet. The supernatant is pipetted out anddiluted in a 1:2 ratio using HBSS. This suspension is centrifuged for 10minutes at a velocity of 600 g. The supernatant is aspirated and thepellet is dissolved in 19 ml of deionized water. After one minute, thepellet is resuspended in 1 ml of 10×PBS. This suspension is centrifugedat 400 g for 10 minutes. The red blood cells are lysed in this processand the remaining cells are mostly neutrophils. The resulting pellet maybe dissolved in media containing BSA in order to avoid the clumping ofcells after a prolonged period of time at room temperature. The celldensity is determined by counting the number of cells using ahemocytometer.

B. Placement of Leukocytes and Leukocyte Migration Mediators in Chamber

20 μl of water are pipetted in the first well of the chamber of thedevice fabricated according to the method disclosed in Section I andmicrocapillary action draws the water into the channel. After ensuringno air bubbles are inside the channel, an additional 10 μl of water arepipetted in the second well of the chamber. After 15 minutes pass andthe hydrostatic pressure equalizes, 10 μl of P-Selectin at aconcentration of 50 μg/mL (obtained from R&D Systems, catalog #ADP3) ispipetted in both wells. The device is incubated for two hours at roomtemperature in a dish with a cover in order to keep the wells fromdrying out. After the incubation, the channel is washed four times using0.1% Bovine Serum Albumin (BSA) in Phosphate Buffer Saline (PBS). Afterthis last wash, all the liquid inside the wells is pipetted out leavingonly liquid in the channel. 20 μl of 0.1% BSA in PBS is added to thefirst well and 10 μl of BSA in PBS is added to the second well. After 15minutes pass and the hydrostatic pressure equalizes, neutrophilsobtained from the method described in part A in 60 μl of media are addedto the first well of each chamber (about 10³ to about 10⁶ cells per wellof a 24 well plate, in volume of 60 μl of media per well) (non-labeledand fluorescently labeled monocytic cell lines-U937 (obtained from ATCC,catalog # TIB-202 and THP-1 (obtained from ATCC, catalog # CRL-1593.2)as well as other primary leukocytes may also be used. As seen in FIG. 7,it is preferred that 40 μl-60 μl of media be used to generate the rangeof flow velocity under normal physiological conditions (about 0.1dynes/cm² to about 20 dynes/cm²).

C. Data Acquisition

Digital images are taken on a Zeiss inverted microscope using AXIOCAM™beginning 15 seconds after the sample comprising leukocytes is added tothe first well. Data is analyzed on AXIOVISION™ software. Time-lapsedimages are taken every 30 seconds for 5 minutes and 15 seconds. 10×objective lens is used to view and record the number of cells rollingalong the channel.

D. Determining the Rolling Velocity of the Leukocytes

In order to characterize the rolling velocity of the leukocytes at aparticular time, an image obtained using the method described in part Cis used measure the distance the leukocytes traveled during the exposuretime of the image. To determine rolling velocity (V), the followingformula is used:V=c(1_(time) /t _(exposure)) where

-   -   c: conversion factor for determining the actual distance the        cells traveled.        This factor may vary from image to image.    -   1_(time): the length of the leukocytes migration in the captured        image.    -   t_(exposure): the exposure time of the image.

Preferably t_(exposure) is 100 milliseconds (ms) when the flow rate isabout 0.1 dynes/cm² to about 20 dynes/cm².

III. Leukocyte Migration Assay Utilizing Device of the Present Inventionwith a Rolling Mediator and Arrest Mediator Disposed in a ChannelTherein

A. Isolation of Leukocytes

Neutrophils are isolated according to the method disclosed in sectionII, part A.

B. Placement of Leukocytes and Leukocyte Migration Mediators in Chamber

20 μl of water are pipetted in the first well of the chamber of thedevice fabricated according to method disclosed in section I.Microcapillary action draws the water into the channel. After ensuringno air bubbles are inside the channel, an additional 10 μl of water arepipetted in the second well of the chamber. After 15 minutes pass andthe hydrostatic pressure equalizes, 10 μl of P-Selectin with aconcentration of 50 μg/mL (obtained from R&D Systems, catalog #ADP3) ispipetted in the first well and 10 μl of ICAM-1 with a concentration of50 μg/mL (obtained from R&D Systems) is simultaneously pipetted in thesecond well. The device is incubated for two hours at room temperaturein a dish with a cover in order to keep the wells from drying out. Afterthe incubation, the channel is washed four times using 0.1% Bovine SerumAlbumin (BSA) in Phosphate Buffer Saline (PBS). After this last wash,all the liquid inside the wells is pipetted out leaving only liquid inthe channel. 20 μl of 0.1% BSA in PBS is added to the first well and 10μl of BSA in PBS is added to the second well. After 15 minutes pass andthe hydrostatic pressure equalizes, neutrophils isolated from part A in60 μl of media are added to the first well of each chamber (about 10³ toabout 10⁶ cells per well of a 24 well plate, in volume of 60 μl of mediaper well) (non-labeled and fluorescently labeled monocytic celllines-U937 and THP-1 as well as primary leukocytes may also be used). Asseen in FIG. 7, it is preferred that 40 μl-60 μl of media be used togenerate the range of flow velocity under normal physiologicalconditions (about 0.1 dynes/cm² to about 20 dynes/cm²).

C. Data Acquisition

Digital images are taken on a Zeiss inverted microscope using AXIOCAM™beginning 15 seconds after the sample comprising leukocytes is added tothe first well. Data is analyzed on AXIOVISION™ software. Time-lapsedimages are taken every 30 seconds for 5 minutes and 15 seconds. 10×objective lens is used to view and record the number of cells rollingalong the channel and adhering to the channel.

IV. Leukocyte Migration Assay Utilizing Confluent Layers of EndothelialCells

A. Isolation of Leukocytes

Neutrophils are isolated according to the method disclosed in sectionII, part A.

B. Placement of Leukocytes and Endothelial Cells in Chamber

10 μL of a 10× dilution of MATRIGEL™ (obtained from BD Bioscience,catalog # 356231) is added to the first well of the device fabricatedaccording to the method disclosed in Section I. 10 μL are added to thefirst well and the microcapillary action draws the solution into thechannel. The MATRIGEL™ is then allowed to gel for about 15 minutes atroom temperature. Another option is to coat the channel with 1 mg/mLconcentration of fibronectin (obtained from GibcoBRL, catalog #33016-015) that is obtained by diluting the stock concentration offibronectin using a 0.1% BSA solution. 5 μL of fibronection at aconcentration of 1 mg/mL are pipetted into the first well andmicrocapillary action draws the solution in to the channel.

Once the channel has been coated with either MATRIGEL™ or fibronectin,the endothelial cells are prepared for seeding. Cells are obtained fromClonetics at Bio-Whittaker in cryogenic vials. They are grown in T75flasks until ready to be split using 0.025% Trypsin/EDTA. The cells areseeded on the channel at a density of 1×10⁵ cells per 5 μl of media perassay for approximately two days to form a confluent monolayer ofendothelial cells. During these two days, the endothelial cells arereplenished with 40 μL of fresh media added into each well. Afterapproximately two days, the endothelial cells are exposed to aconcentration of 1 ng/ml of TNF-α (other chemokines may alternatively beused) for a period of four hours at 37° C. At the end of the four hours,the TNF-α is washed using 60 μL of fresh media twice. The volume ofmedia inside each well is replaced with 15 μL of fresh media.Neutrophils isolated from Section II, part A in 60 μl of media are addedto the first well of chamber the (about 10³ to about 10⁶ cells per wellof a 24 well plate, in volume of 60 μl of media per well) (non-labeledand fluorescently labeled monocytic cell lines-U937 and THP-1 as well asprimary leukocytes may also be used.) If a monocytic cell line is used,the cells are fluorescence labeled using cell tracker probes (obtainedfrom Molecular Probes, catalog #s C-2925 and C-2927). The cells areincubated with a 1 μM concentration of probes for 30 minutes at 37° C.The media is then changed and the cells are placed inside an incubatorfor an additional 30 minutes.

As seen in FIG. 7, it is preferred that 40 μL-60 μL of media be used togenerate the range of flow velocity under normal physiologicalconditions (about 0.1 dynes/cm² to about 20 dynes/cm²).

C. Data Acquisition

Digital images are taken on a Zeiss inverted microscope using AXIOCAM™beginning 15 seconds after the sample comprising leukocytes is added tothe first well. Data is analyzed on AXIOVISION™ software. Time-lapsedimages are taken every 30 seconds for 5 minutes and 15 seconds. 10×objective lens is used to view and record the number of cells rollingalong the channel.

D. Determining the Rolling Velocity of the Leukocytes

In order to characterize the rolling velocity of the cells at aparticular time, an image obtained from the method described in part Cis used to measure the distance the leukocytes traveled during theexposure time of the image. To determine rolling velocity (V), thefollowing formula is used:V=c(1_(time) /t _(exposure)) where

-   -   c: conversion factor for determining the actual distance the        cells traveled.        This factor may vary from image to image.    -   1_(time): the length of the leukocytes migration in the captured        image.    -   t_(exposure): the exposure time of the image.

Preferably t_(exposure) is 100 ms when the flow rate is about 0.1dynes/cm² to about 20 dynes/cm².

V. Inhibition of Leukocyte Migration Assay Utilizing Device of thePresent Invention With a Rolling Mediator and an Arrest MediatorDisposed in a Channel Therein

A. Isolation of Leukocytes

Neutrophils are isolated according to the method disclosed in sectionII, part A.

B. Placement of Leukocytes, P-selectin, and P-selectin Antibodies in theChamber

With respect to five chambers, 20 μl of 0.1% BSA are pipetted in thefirst well of each chamber of the device fabricated according to themethod described in Section I. Microcapillary action draws water intothe channels. After ensuring no air bubbles are inside the channels, anadditional 10 μl of BSA are pipetted in the second well of each chamber.After 15 minutes pass and the hydrostatic pressure equalizes, 10 μl ofP-Selectin (50 μg/mL) are pipetted in first wells and 10 μl of ICAM-1(50 μg/mL) are pipetted into the second wells using a multipipettor. Thedevice is incubated for two hours at room temperature in a dish with acover in order to keep the wells from drying out. After the incubation,the channels of each well are washed four times using 0.1% Bovine SerumAlbumin (BSA) in Phosphate Buffer Saline (PBS). With respect to the fivedifferent chambers, 100 ng/mL of P-selectin antibody is pipetted intothe first well of chamber #1; 10 ng/mL of P-selectin antibody ispipetted into first well of chamber #2; and 1 ng/mL of P-selectinantibody is pipetted into the first well of chamber #3; 100 μg/mL ofP-selectin antibody is pipetted into the first well of chamber #4; and0.1% BSA in PBS is pipetted into the first well of chamber #5. Thedevice is incubated for thirty minutes at room temperature in a dishwith a cover in order to keep the wells from drying out. Afterincubation, the channels are washed first with 20 μl of BSA, then with10 μl of BSA and then 0.1% BSA in PBS. Neutrophils in 20 μl of media areadded to the first well of each chamber (about 10³ to about 10⁶ per wellof a 24 well plate, in volume of 20 μl of media per well) (non-labeledand fluorescently labeled monocytic cell lines-U937 and THP-1 as well asprimary leukocytes may be used). Digital images are taken on a Zeissinverted microscope using AXIOCAM™ beginning 15 seconds after the samplecomprising leukocytes is added to the first well. Data is analyzed onAXIOVISION™ software. Time-lapsed images are taken every 30 seconds for5 minutes and 15 seconds. 10× objective lens is used to view and recordthe number of cells rolling after the treatment with P-selectinantibody. As seen from FIGS. 8 and 9, a 100 ng/mL dilution of theantibody is a preferred concentration to inhibit the rolling of thecells. As seen from the still photo images of FIG. 9, the number ofleukocytes that roll and adhere to the endothelium are reduced in thepresence of anti-P selectin.

C. Placement of Leukocytes, E-Selectin, and E-Selectin Antibodies in theChamber

With respect to five chambers, 20 μl of 0.1% BSA are pipetted in thefirst well of each chamber of the device fabricated according to themethod described in Section I. Microcapillary action draws the BSA intothe channels. After ensuring no air bubbles are inside the channels, anadditional 10 μl of 0.1% BSA are pipetted in the second well of eachchamber. After 15 minutes pass and the hydrostatic pressure equalizes,10 μl of E-Selectin (50 μg/mL) are pipetted in the first wells and 10 μlof ICAM-1 (50 μg/mL) are pipetted into the second wells using amultipipettor. The device is incubated for two hours at room temperaturein a dish with a cover in order to keep the wells from drying out. Afterthe incubation, the channels of each well are washed four times using0.1% Bovine Serum Albumin (BSA) in Phosphate Buffer Saline (PBS). Withrespect to the five different chambers, 100 ng/mL of E-selectin antibodyis pipetted into the first well of chamber #1; 10 ng/mL of E-selectinantibody is pipetted into first well of chamber #2; and 1 ng/mL ofE-selectin antibody is pipetted into the first well of chamber #3; 100μg/mL of E-selectin antibody is pipetted into the first well of chamber#4; and 0.1% BSA in PBS is pipetted into the first well of chamber #5.The device is incubated for thirty minutes at room temperature in a dishwith a cover in order to keep the wells from drying out. Afterincubation, the channels are washed four times with 0.1% BSA in PBS.Neutrophils in 20 μl of media are added to the first well of eachchamber (about 10³ to about 10⁶ cells per well of a 24 well plate, involume of 20 μl of media per well) (non-labeled and fluorescentlylabeled monocytic cell lines-U937 and THP-1 as well as primaryleukocytes may be used). Digital images are taken on a Zeiss invertedmicroscope using AXIOCAM™ beginning 15 seconds after the samplecomprising leukocytes is added to the first well. Data is analyzed onAXIOVISION™ software. Time-lapsed images are taken every 30 seconds for5 minutes and 15 seconds. 10× objective lens is used to view and recordthe number of cells rolling after the treatment with E-selectinantibody. As seen from FIG. 10, a 100 ng/mL dilution of the antibody isa preferred concentration to inhibit the rolling of the cells. As seenfrom the still photo images of FIG. 10, the number of leukocytes thatroll and adhere to the endothelium are reduced in the presence of anti-Eselectin.

VI. Inhibition of Leukocyte Migration Assay Utilizing Device of thePresent Invention with Confluent Layers of Endothelial Cells Disposed ina Channel Therein

A. Isolation of Leukocytes

Neutrophils are isolated according to the method disclosed in sectionII, part A.

B. Placement of Leukocytes and Endothelial Cells in Chamber

Endothelial cells are placed and activated in four different channels offour chambers (#1-#4) according to the method disclosed in section IV,part B. With respect to a fifth (#5) chamber, endothelial cells areplaced in the channel, but are not activated. With respect to these fivedifferent chambers, 100 μg/ml of P-selectin antibody is pipetted intothe first well of chamber #1; 100 μg/ml of E-selectin antibody ispipetted into the first well of chamber #2; 100 μg/ml of VCAM-1 antibodyis pipetted into the first well of chamber #3; and 100 μg/ml of BSA inPBS is pipetted into the first well of chamber #4. The device isincubated for thirty minutes at room temperature in a dish with a coverin order to keep the wells from drying out. After incubation, thechannels are washed four times with 0.1% BSA in PBS. Neutrophils in 20μl of media are added to the first well of each chamber (about 10³ toabout 10⁶ cells per well of a 24 well plate, in volume of 20 μl of mediaper well) (non-labeled and fluorescently labeled monocytic celllines-U937 and THP-1 as well as primary leukocytes may be used). Digitalimages are taken on a Zeiss inverted microscope using AXIOCAM™ beginning15 seconds after the sample comprising leukocytes is added to the firstwells. Data is analyzed on AXIOVISION™ software. Time-lapsed images aretaken every 30 seconds for 5 minutes and 15 seconds. 10× objective lensis used to view and record the number of cells rolling after thetreatment with the antibodies as seen in FIG. 10.

The foregoing description and examples have been set forth merely toillustrate the invention and are not intended to be limiting. Sincemodifications of the disclosed embodiments incorporating the spirit andsubstance of the invention may occur to persons skilled in the art, theinvention should be construed to include everything within the scope ofthe appended claims and equivalents thereof.

Example VII Selective Activation of Endothelial Cells by Delivery ofTNF-α in a Gradient Created by Laminar Flow

The surface of a device of the present invention was coated withendothelial cells and allowed to grow to confluence (to create a “lawn”of cells). TNF-α was delivered to the lawn of endothelial cells vialaminar flow to “activate” the endothelial cells. Each stream ofsolutions containing TNF-α were at different concentrations, thuscreating a gradient perpendicular to the channel. This gradienteffectively delivered TNF-α to the lawn of endothelial cells atdifferent concentrations at different positions on the lawn of cells.Leukocytes were then flowed over the lawn of activated endothelialcells. Referring to FIG. 12, only those endothelial cells that wereactivated by TNF-α provide suitable “attachment” sites for theleukocytes. The leukocytes did not attach equally to the entire lawn,but attached to the areas of the endothelial cell lawn that had beenexposed to high concentrations of TNF-α and did not attach to thoseareas of the lawn that had been exposed to low concentrations of TNF-α,or those areas not exposed to TNF-α at all. These results indicate thatthere was indeed a creation of a concentration gradient of TNF-α by thelaminar flow.

1. A system for monitoring leukocyte migration comprising: a deviceincluding a housing having a base member and a top member defining aplurality of chambers therein, each of the plurality of chambersincluding: a first well region including at least one first well; asecond well region including at least one second well; and a channelregion including at least one channel connecting the first well regionand the second well region with one another, wherein the at least onechannel is formed as a through-hole in the top member; a first fluidstream having a first concentration of a first substance; and a secondfluid stream having a second concentration of a second substance,wherein the first and second concentrations are different from oneanother and the first and second fluid streams are in fluidcommunication with at least one of the plurality of chambers, andwherein the first fluid stream and the second fluid stream flow adjacentand parallel to each other without mixing, to create a dynamicconcentration gradient.
 2. The system of claim 1, wherein the first andsecond substance are the same.
 3. The system of claim 1, wherein thefirst and second substance are different.
 4. The system of claim 1,wherein the first fluid stream and the second fluid stream converge intoa single third fluid stream that is in fluid communication with the atleast one of the plurality of chambers, wherein the third fluid streamcomprises a concentration gradient of the first and second substances,the concentration gradient being substantially perpendicular to thedirection of flow of the third fluid stream.
 5. The system of claim 1,wherein the first and second fluid streams converge into a single thirdstream, then diverge into three separate fourth, fifth, and sixthstreams, and then re-converge into a single seventh stream, the seventhstream in fluid communication with the at least one of the plurality ofchambers under conditions of substantially laminar flow.
 6. The systemof claim 1, wherein at least one of the first and second substance is acytokine.
 7. The system of claim 6, wherein the cytokine is tumornecrosis TNF-α.
 8. The system of claim 1, wherein at least one of thefirst and second substance is a test agent.
 9. A method of monitoringleukocyte migration comprising: providing a device including a housinghaving a base member and a top member defining a plurality of chamberstherein, each of the plurality of chambers including: a first wellregion including at least one first well; a second well region includingat least one second well; and a channel region including at least onechannel connecting the first well region and the second well region withone another, wherein the at least one channel is formed as athrough-hole in the top member; disposing at least one leukocytemigration mediator, or endothelial cells in the at least one channel;delivering a sample comprising leukocytes to the at least one channel bylaminar flow; and observing the interaction between the leukocytes andthe at least one leukocyte migration mediator or the interaction betweenthe leukocytes and the endothelial cells.
 10. The method of claim 9,further comprising providing a video camera operatively linked to thedevice for viewing the at least one channel.
 11. The method of claim 10,wherein the video camera is adapted to capture an image duringpredetermined intervals over a predetermined period of time.